Automated GC-MS Determination of Δ9-Tetrahydrocannabinol, Cannabinol and Cannabidiol in Hair.

The determination of Δ(9)-tetrahydrocannabinol (THC), cannabinol (CBN) and cannabidiol (CBD) in hair is a major routine task in forensic laboratories worldwide. A comprehensively automated liquid-liquid extraction (LLE) method has been developed. The automation was carried out by an x-y-z sample robot equipped with modules capable of shaking, centrifugation and solvent evaporation. It comprises digestion of hair in sodium hydroxide solution, LLE, extract evaporation, reconstitution in silylation reagent, inlet derivatization and GC-MS analysis. Method validation guidelines of the Society for Toxicological and Forensic Chemistry were fulfilled. The limit of quantification (LOQ) was 0.01 ng/mg for THC, 0.06 ng/mg for CBN and 0.03 ng/mg for CBD. This is below the required LOQ for THC (0.02 ng/mg) in medical psychological assessments in Germany. Also it is far below the required LOQ of the Society of Hair Testing of 0.1 ng/mg for THC. Four-round robin tests were passed successfully and several post- and ante-mortem samples were analyzed. To date the method is routinely employed at the Institute of Legal Medicine in Giessen, Germany. To the best of our knowledge, this is the first publication on a comprehensively automated classical LLE workflow in the field of hair analysis.

Development and validation of an automated liquid-liquid extraction GC/MS method for the determination of THC, 11-OH-THC, and free THC-carboxylic acid (THC-COOH) from blood serum.

The analysis of Δ(9)-tetrahydrocannabinol (THC) and its metabolites 11-hydroxy-Δ(9)-tetrahydrocannabinol (11-OH-THC), and 11-nor-9-carboxy-Δ(9)-tetrahydrocannabinol (THC-COOH) from blood serum is a routine task in forensic toxicology laboratories. For examination of consumption habits, the concentration of the phase I metabolite THC-COOH is used. Recommendations for interpretation of analysis values in medical-psychological assessments (regranting of driver's licenses, Germany) include threshold values for the free, unconjugated THC-COOH. Using a fully automated two-step liquid-liquid extraction, THC, 11-OH-THC, and free, unconjugated THC-COOH were extracted from blood serum, silylated with N-methyl-N-(trimethylsilyl) trifluoroacetamide (MSTFA), and analyzed by GC/MS. The automation was carried out by an x-y-z sample robot equipped with modules for shaking, centrifugation, and solvent evaporation. This method was based on a previously developed manual sample preparation method. Validation guidelines of the Society of Toxicological and Forensic Chemistry (GTFCh) were fulfilled for both methods, at which the focus of this article is the automated one. Limits of detection and quantification for THC were 0.3 and 0.6 µg/L, for 11-OH-THC were 0.1 and 0.8 µg/L, and for THC-COOH were 0.3 and 1.1 µg/L, when extracting only 0.5 mL of blood serum. Therefore, the required limit of quantification for THC of 1 µg/L in driving under the influence of cannabis cases in Germany (and other countries) can be reached and the method can be employed in that context. Real and external control samples were analyzed, and a round robin test was passed successfully. To date, the method is employed in the Institute of Legal Medicine in Giessen, Germany, in daily routine. Automation helps in avoiding errors during sample preparation and reduces the workload of the laboratory personnel. Due to its flexibility, the analysis system can be employed for other liquid-liquid extractions as well. To the best of our knowledge, this is the first publication on a comprehensively automated classical liquid-liquid extraction workflow in the field of forensic toxicological analysis. Graphical abstract GC/MS with MPS Dual Head at the Institute of Legal Medicine, Giessen, Germany. Modules from left to right: (quick) Mix (for LLE), wash station, tray 1 (vials for extracts), solvent reservoir, (m) VAP (for extract evaporation), Solvent Filling Station (solvent supply), cooled tray 2 (vials for serum samples), and centrifuge (for phase separation).

[Intentional poisoning of two wives by their husband?]. / Vorsätzliche Vergiftung zweier Frauen durch ihren Ehemann?

The authors report on the death of two women who were married to the same man one after the other. Exhumation and toxicological investigation of the first wife, who had died 7 years before, did not produce any conclusive evidence of homicide. With regard to the circumstances of death of the second wife the husband made different statements. According to the result of the chemical and toxicological investigations death was caused by acute intoxication with the beta-blocker metoprolol. The man was found guilty of killing on request (which is a criminal offence in Germany) by administering the beta-blocker metoprolol through a transnasal gastric tube.

Quantitative determination of 43 common drugs and drugs of abuse in human serum by HPLC-MS/MS.

An analytical procedure for the simultaneous determination in human serum of 43 common drugs of abuse and their metabolites belonging to the different chemical and toxicological classes of amphetamines, benzodiazepines, dibenzazepines, cocaine, lysergic acid diethylamide, opioids, phencyclidine, tricyclic antidepressants, and zolpidem, using 33 deuterated standards, is presented. The sample treatment was developed to be a very simple protein precipitation and filtration. All analyses were performed with a high-performance liquid chromatography-electrospray ionization-tandem mass spectrometry in positive ionization mode. All analytes were calibrated up to 550 µg/L. The limit of detection ranged from 0.6 ng/mL (EDDP) to 13.7 ng/mL (flunitrazepam). The method has been validated according to the guidelines of the Gesellschaft für Toxikologische und Forensische Chemie, using three multiple reaction mode (MRM) transitions and retention time for positive compound identification, instead of two MRMs, in anticipation of the new guidelines for January 2011.

Accidental fatal ingestion of colchicine-containing leaves--toxicological and histological findings.

A 65 year old woman had collected some plant leaves, cooked them in water and ate this mash with honey. A few hours after consumption first symptoms of intoxication like sickness, stomach aches and diarrhoea appeared. Her condition deteriorated, she was admitted to hospital, became comatose and finally died due to a multiorgan failure. The plants she had collected were identified as autumn crocus (Colchicum autumnale). Colchicine-concentrations detected in blood samples (HPLC/DAD) taken in hospital approximately 24 h after ingestion were 50 microg/l, in post-mortem femoral blood 65 microg/l colchicine were found (toxic range: >24 microg/l). Also all tested organs contained equally high colchicine levels. Histological examination of the liver showed diffuse vacuolization in the cytoplasma of hepatocytes. Possibly due to the relatively short survival time, only isolated mitotic structures were found within the epithelium of the colon.

Oxalate-crystals in different tissues following intoxication with ethylene glycol: three case reports.

Three alcoholics (62 years, 47 years, 83 years) died between 11 and 18 h after ingestion of ethylene glycol (EG). One person committed suicide. Observed symptoms of intoxication were seizures, respiratory depression, arrhythmias and hypotonia. All died in hospital after failed attempts at resuscitation, one person did so after an 11h dialysis treatment. EG was detected in blood in concentrations of between 1 and 3mg/L (toxic range: >0.3mg/L). One case presented a blood alcohol concentration (bac) of 1.14 per thousand. Further toxic substances were not found. Using special staining techniques, oxalate crystals were found in samples from the kidneys, explaining renal failure, and in the medial layer of cerebral vessels.

[Investigations concerning the threshold value between endogenous and exogenous GHB (liquid ecstasy)]. / Untersuchungen zum Grenzwert zwischen endogener und exogener gamma-hydroxybuttersäure (GHB/Liquid Ecstasy).

The article describes critical investigations concerning the threshold value between endogenous and exogenous concentrations of gamma-hydroxybutyric acid (GHB/"liquid ecstasy") in human blood. The values of GHB in the blood samples of 50 blood donors and 50 postmortem cases were measured with a validated gas-chromatographic/mass-spectrometric procedure according to the guidelines of the GTFCh (Society of Toxicological and Forensic Chemistry). GHB-concentrations were found to range between 0.11 and 1.56 mg/L (mean value 0.54 mg/L/standard deviation 0.37 mg/L/coefficient of variation 68.4 %) in the donors' blood, and between 2.2 and 116 mg/L (mean value 32.4 mg/L/standard deviation 25.6 mg/L/coefficient of variation 79 %) in the postmortem samples, respectively.

Reliability of the cloned-enzyme donor immunoassay (CEDIA) for cocaine in human serum in the range between the detection limit and the cut-off.

Immunoassays are used worldwide for the rapid screening of drugs. Despite the fact that they are highly valuable tools for the testing of legal and illicit drugs, there is a non-negligable risk of false-positive and false-negative findings and many pitfalls must be taken into account when using these tests in an uncritical manner and without valid confirmation procedures. In order to check the correlation between cloned-enzyme donor immunoassay (CEDIA) readings and exact determined gas chromatography/mass spectrometry (GC/MS) values for benzoylecgonine, a total of 472 serum samples was measured with an immunoassay (CEDIA) as well as GC/MS. As a result, it was shown that in the lower area of concentration, up to approx. 300 ng equ. benzoylecgonine/ml, there is a semiquantitative useful correlation. With higher concentrations, the values shift more and more apart and many runaways are visible. Generally the use of tests for materials other than those declared is excluded from the manufacturer's warranty and is at the user's own risk, but the authors found that many immunochemical tests with a certification only for the use in urine are well suited to the screening of serum samples, and even low readings could be confirmed by GC/MS. With blood or serum as sample, it could be regularly observed that generally very small readings could not only be confirmed with mass spectrometry but could in most cases also be quantified. This was the key factor in determining the rate of confirmation in 495 cases. In total, an incredibly high reliability for the CEDIA cocaine test (via benzoylecgonine) was shown up to the area of detection (detection limit), far below the cut-off. These results confirm the extraordinary sensitivity and specificity of this test also for blood or serum as sample material to be tested.

[Drug screening in urine--common pitfalls in measurement and interpretation]. / Drogenscreening im Harn--häaufige Fehlerquellen bei der Messung und Interpretation.

Nowadays immunoassays are used for the rapid screening of drugs world-wide. Despite the fact that they are a highly valuable tool for the test of legal and illicit drugs, there is a non-negligible risk of false positive and false negative findings, and many pitfalls must be taken into account when using these tests in an uncritical manner and without valid confirmation procedures. In accordance with the committee for "problems of threshold values for medical and addictive drugs" set up by German scientific societies (in particular GTFCh, DGRM and DGVM), immunoassays are recommended as a useful tool for screening. But confirmation analysis with conclusive methods (GC/MS or LC/MS) is indispensable for valid substance identification, discrimination between active and inactive metabolites, detection of escort substances and accurate determination of concentrations in body fluids.

Immunoassays for drug screening in urine : Chances, challenges, and pitfalls.

Immunoassays are presently used worldwide for the rapid screening of drugs. Despite the fact that they are a highly valuable tool for the testing of legal and illicit drugs, there is a real risk of false-positive and false-negative findings and many pitfalls must be taken into account when these tests are used in an uncritical manner and without valid confirmation procedures. In a long-standing successful cooperation with a large psychiatric hospital, we checked doubtful and nonplausible immunochemical findings in urine with gas chromatography-mass spectrometry (GC-MS) confirmation methods. The reported case histories demonstrate typical pitfalls, for example, influence of changing nutritional habits, use of sweeteners (cyclamate), uncritical creatinine correction, impaired elimination, or cross-reactivities of simultaneous therapeutic medication. In accordance with national and international associations, immunoassays are recommended as a useful tool for screening. However, confirmation analysis with conclusive methods (GC-MS or liquid chromatography-MS) is unavoidable for valid substance identification, discrimination between active and inactive metabolites, detection of congeners, and accurate determination of concentrations in body fluids.

[Reliability of % vol. declarations on labels of wine bottles]. / Zur Verlässlichkeit von Vol.-%-Angaben auf Weinflaschenetiketten.

The Council Regulation (EC) no. 1493/1999 of 17 May 1999 on the common organisation of the market in wine (Abl. L 179 dated 14/7/1999) and the GMO Wine 2000 (Annex VII A) stipulates that the labels of wine bottles have to indicate, among others, information on the sales designation of the product, the nominal volume and the alcoholic strength. The latter must not differ by more than 0.5% vol. from the alcoholic strength as established by analysis. Only when quality wines are stored in bottles for more than three years, the accepted tolerance limits are +/- 0.8% vol. The presented investigation results show that deviations have to be taken into account which may be highly relevant for forensic practice.

Pitfalls of toxicological analysis.

Pitfalls are a permanent risk in all fields of forensic- and clinical-toxicological analysis and are not only concerned with immunoassays, although they are at present mostly discussed with regard to this subject. There are risks inherent in the field of preanalysis (e.g. the sampling, transportation, storage und treatment of the samples immediately prior to analysis). Furthermore, various pitfalls during measurement itself are to be avoided in connection with derivatization, choice of analysis parameters, memory-effects and many other sources of error. Another important section concerns the pharmacokinetic and pharmacodynamic interpretation of analytical results. Many toxicokinetic (e.g. enzyme induction) and toxicodynamic factors (e.g. tolerance effects) must be considered with special regard to plausibility controls (e.g. constellation, longitudinal, transversal and trend monitoring).

[Immunochemical screening: manipulation, handling and interpretation errors, exemplified by benzodiazepines]. / Immunchemisches Screening: Manipulationen, Störungen und Fehlinterpretationen, dargestellt am Beispiel der Benzodiazepine.

The article describes problems related to immunochemical screening-tests with special regard to benzodiazepines, which are commonly used for therapeutic purposes. Focal points are adulteration, manipulation and handling of samples, the selection of cut-off-values, specificity and cross-reactivity, influence of biotransformation and frequently observed pitfalls in the interpretation of the analytical results.

[Screening and detection of amphetamine derivatives in biological stains]. / Screening und Nachweis von Amphetaminderivaten in biologischem Spurenmaterial.

A GC/MS-method is described for the screening, detection and determination of the commonly used drugs amphetamine, methamphetamine, MDA, MDMA and MBDB in small blood samples and bloodstains using solid phase SPE columns and a pipetting robot (Gilson Aspec XL). The detection limits are in the order of 0.03 to 0.08 microgram/L and the correlation factors between 0.9982 and 0.9998. Furthermore the stability was investigated covering a storage time of 64 days. The method has proven useful in forensic cases with only small sample volumes or bloodstains.

Studies of interferences of apomorphine and its metabolites or decomposition products with immunochemical screening tests for legal and illicit drugs after therapeutic doses of apomorphine.

This study deals with the question of whether apomorphine (CAS 314-19-2; e.g. in Ixense) and its metabolites or decomposition products interfere with the specificity of immunochemical screening tests (immunoassays) for legal and illicit drugs. It was the result of the investigations that after the therapeutic use of apomorphine no relevant false-positive screening findings were observed with the CEDIA (cloned enzyme donor immuno assay) and FPIA (fluorescence polarisation immuno assay) tests, which are commonly encountered in drug-screening programs, when using recommended cut-off values for urine.

Simultaneous screening and detection of drugs in small blood samples and bloodstains.

A gas chromatography-mass spectrometry (GC-MS) method is described for the screening and detection of morphine, codeine, cocaine, benzoylecgonine, methylecgonine, cocaethylene, delta-9-tetrahydrocannabinol (THC), 11-nor-9-carboxy-THC (THC-COOH), 11-hydroxy-THC (11-OH-THC), amphetamine, methamphetamine, 3,4-methylenedioxyamphetamine (MDA), 3,4-methylenedioxymetamphetamine (MDMA) and N-methyl-1-(3,4-methylenedioxyphenyl)-2-butanamine (MBDB) in small blood samples and bloodstains using solid phase SPE columns and a pipetting robot (Gilson Aspec XL). The detection limits are in the order of 1.62-4.10 ng/50 microl spot (amphetamines), 0.15-0.82 ng/50 microl spot (cannabinoids), 1.67-4.70 ng/50 microl spot (cocaine and derivatives) and 4.53-4.91 ng/50 microl spot (opiates) and the correlation factors are between 0.9957 and 0.9999. The method has proven useful in forensic cases with only small sample volumes or bloodstains.